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Functional elucidation of human somatostatin receptor-3 : implications in breast tumor biology War, Sajad Amin


Somatostatin (SST) inhibits cell proliferation through five SST receptors (SSTR1-5). Amongst all SSTR subtypes, SSTR2 and SSTR3 are the prominent receptor subtypes which exert antiproliferative effects in cells of different origin. SSTR2-mediated inhibition of cell proliferation is largely cytostatic, whereas SSTR3 is cytotoxic. Whether SSTR2/SSTR3 display synergistic antiproliferation than single receptor is not well understood. To ascertain the role of SSTR3, the present study was first conducted in HEK-293 cells which lack endogenous SSTRs expression. Cells were stably transfected with wt-SSTR3, treated with agonist and studied for dimerization, cAMP, receptor trafficking and signaling molecules. Since receptor signaling properties are confined in C-tail, cells expressing C-tail deleted SSTR3 were also studied for comparative analysis. wt-SSTR3 exists as preformed homodimer at cell surface and displays agonist-mediated cytotoxic effects. The cell surface expression, homodimerization and agonist-induced internalization of SSTR3 were independent of C-tail, whereas agonist-mediated apoptosis was lost upon Ctail deletion. Next, HEK-293 cells cotransfected with SSTR2/SSTR3 were examined for heterodimerization and signaling molecules governing cell proliferation. Pb- FRET/CO-IP analyses suggest SSTR2/SSTR3 heterodimerization. The decreased cAMP upon agonist activation of SSTR2/SSTR3 suggests that this heterodimer is functional. Agonist-mediated SSTR2/SSTR3 antiproliferation was Gi-dependent, and involved apoptosis and cell-cycle arrest. iii To derive direct pathological significance of the observations from heterologous system, additional experiments were conducted in two breast cancer cell lines MCF-7 and MDA-MB-231, which differ in origin and biochemical features including presence or absence of ERα. Breast tumor cell lines overexpressing SSTR3 were studied for cell proliferation and downstream signaling molecules. EGF served as an index of positive cell proliferation. SSTR3 overexpression in MCF-7 (R3-MCF-7) and MDA-MB-231 (R3-MB-231) cells displayed inhibition of EGF-induced proliferation and enhanced antiproliferative effect of SSTR3-specific agonist in comparison to non-transfected cells. SSTR3 overexpression in R3-MCF-7 cells constitutively enhanced TUNEL staining, PARP-1 and p27Kip1 expression suggesting apoptosis and cell-cycle arrest. Conversely, in R3-MB-231 cells, SSTR3 overexpression exerted cytostatic but not cytotoxic effects. These results provide compelling evidence for antiproliferative role of SSTR3 in breast cancer cell lines. The constitutive activation of cytotoxic signaling in R3-MCF-7 but not R3-MB-231 cells reveals a distinct cell-specific role for SSTR3 in breast tumor biology.

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